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Normal Phase Chromatography
Normal-phase chromatography is the original form of liquid chromatography, as it was used in the early years of the discipline. It is sometimes also referred to as adsorption chromatography when, due to the choice of stationary phase, the separation mechanism is based on adsorption. Normal-phase chromatography is generally characterised by stationary phases with polar surface properties and a mobile phase of a non-polar nature.
Technical Data
Basics
The stationary phases of normal phase chromatography
Unmodified silica gels or aluminium oxide particles are often used as stationary phases, as the separation mechanism in these materials is primarily adsorption; consequently, this can be referred to as ‘true’ adsorption chromatography.
Other materials suitable for use in normal-phase mode (i.e. with non-polar mobile phases) include specially modified silica gels (cyano, amino, diol, nitro); however, these will not be discussed in detail here. More detailed information on this can be found under the heading ‘Medium-polar phases’.
Mobile phases of normal phase chromatography
Almost all common organic solvents and their mixtures can be used as mobile phases in normal phase chromatography, as long as they are not strong acids or bases. Aliphatic hydrocarbons, such as hexane or heptane, have the weakest elution power. As the polarity of the solvent increases, so does its elution power. Polar solvents used include acetic acid ethyl ester, acetone, THF or various aliphatic alcohols.
What is the separation in normal phase chromatography based on?
Due to the ability of the stationary phase to form hydrogen bonds, electrostatic and other intermolecular interactions with the analytes, retention in normal phase chromatography is based on the different degrees of adsorption of the analytes on the stationary phase. Polar analytes are therefore retained more strongly than non-polar analytes, as they are more able to enter into polar interactions with the stationary phase.
How do I select the correct normal phase column?
Polar stationary phases are used in normal phase chromatography. The simplest phase is pure silica. The interaction is based on adsorption. However, the disadvantage of pure silica phases is that small amounts of water in the mobile phase can accumulate on the stationary phase and thus change the retention and selectivity. Therefore, the reproducible operation of pure silica columns is often difficult. In addition, a long equilibration period is often required to operate the columns from pure silica. Other possibilities are Medium polar phases such as cyano, amino or diol, which can also be used in normal phase chromatography.
The different phases for the normal phase initially differ in their polarity: Silica > Amino > Diol > Cyano
There are also special applications in which different phases are used:
- Silica: separation of very non-polar compounds (carotenoids or lipids), separation of extremely polar compounds (are not retarded in RP), separation of isomers (achiral), use in the DC and in flash chromatography
- Amino: fat-soluble vitamins, alternative selectivity to silica
- Diol: steroids, alternative selectivity for hydrogen bonds and usually longer retention compared to silica
- Cyano: steroids, different selectivity for double bonds, non-polar phase for normal phase chromatography
Other manufacturers of normal phase chromatography columns:
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Agela Technologies
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Agilent Technologies
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Avantor-ACE
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Bischoff
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Dikma
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Dr. Maisch
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GL Sciences
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Hamilton
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Imtakt
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Macherey-Nagel
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Merck
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Merck Supelco (Sigma-Aldrich)
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MicroSolv
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Nacalai Tesque
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Nouryon - Kromasil
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Osaka Soda (Shiseido)
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Perkin-Elmer
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Restek
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Sepax
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Shodex (Resonac)
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SIELC
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SMT-Separation Methods Technologies
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Sumika Chemicals
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Thermo Scientific
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Trajan Scientific (SGE)
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Waters
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Welch Materials
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